Successful gene therapy depends on the efficient delivery to and expression of genetic information within the cells of a living organism. Most delivery mechanisms used so far involve viral vectors. Viruses have developed diverse and highly sophisticated mechanisms to achieve this goal including crossing of the cellular membrane, escape from lysosomal degradation, delivery of their genome to the nucleus and, consequently, have been used in many gene delivery applications.
Non-viral vectors, which are based on receptor-mediated mechanisms (Perales et al., Eur. J. Biochem. 226:255-266, 1994; Wagner et al., Advanced Drug Delivery Reviews 14:113-135, 1994) or on lipid-mediated transfection (Eelgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 1987; Behr et al., Proc. Natl. Acad. Sci. U.S.A. 86:6982-6986, 1989; Gao et al., Biochem. Biophys. Res. Communic. 179:280-285, 1991; Behr, Bioconjugate Chemistry 5:382-389, 1994; Fahrhood et al., Annals New York Academy of Sciences, 716:23-35, 1994; Ledley, Human Gene Therapy 6:1129-1144, 1995) promise to have advantages with respect to large--scale production, reduced risks related to viral vectors, targeting of transfectable cells, lower immunogenicity and the capacity to deliver larger fragments of DNA.
The development of non-viral vectors for the delivery of nucleic acids into cells necessitates molecules that can associate with nucleic acids to allow these large, hydrophilic polyanions to cross the cell membrane, which is a hydrophobic, negatively charged barrier of a phospholipid bilayer, help them to escape from lysosomal degradation and facilitate their entry into the nucleus. Expression of the gene of interest also depends on the accessibility of the delivered nucleic acid to the cellular transcription machinery, most likely necessitating dissociation of the complexes.
Although having been described as early as 1965 (Bangham et al., J. Mol. Biol. 13:238-252, 1965), liposomes, which can encapsulate molecules of interest for delivery to -he cell, did not reach the marketplace as injectable therapeutics in humans until the 1990s (Gregoriadis, Trends in Biotechnol. 13:527-537, 1995). This delay is attributable to difficulties in obtaining reproducible formulations with acceptable stabilities. A major breakthrough was the development of "sterically stabilized (stealth) liposomes" which contained a certain percentage of polyethyleneglycol (PEG)-modified phospholipids (PEG-PLs) (Gregoriadis, Trends in Biotechnol. 13:527-537, 1995; Lasic, Angew. Chem. Int. Ed. Engl. 33:1685-1698, 1994). These PEG-PL containing liposomes proved to be more successful in evading detection and elimination by the reticulo-endothelial system, which resulted in greatly enhanced circulation half-lives. While (PEG)-modified liposomes have been most widely used, other hydrophilic polymers such as poly(vinyl pyrrolidone) which augment the stability of liposomes when grafted on their surface have been described (Torchilin, V. P. et al., Biochim. Biophys. Acta 1195:181-184, 1994).
Progress in lipid-mediated nucleic acid transfer into cells was advanced with the introduction of cationic lipids as vehicles for the transfer of nucleic acids into cells (Felgner et al., Proc. Natl. Acad. Sci. 84:7413-7417, 1987; Behr et al., Proc. Natl. Acad. Sci. 86:6982-6986, 1989; Gao et al., Biochem. Biophys. Res. Communic. 179:280-285, 1991; Behr, Bioconjugate Chemistry 5:382-389, 1994; Fahrhood et al., Annals New York Academy of Sciences 716:23-35, 1994; Ledley, Human Gene Therapy 6:1129-1144, 1995). Because cationic lipids are positively charged, they are able to complex with negatively charged nucleic acids. Unlike liposomes, these complexes do not require an encapsulation step and are prepared by simple mixing of components. The complexes essentially comprise of lipid-coated nucleic acid, in which the positively charged coat of the complex neutralizes the negative charges of the nucleic acid and, also, can efficiently bind to the negatively charged cell surface, facilitating entry of nucleic acid into the cell (Farhood et al., Annals N.Y. Acad. Sci. 716:23-35, 1994).
The advantages of using cationic lipids to mediate transfection of nucleic acids include the simplicity of preparation of the complexes, the ability of the lipid component to complex most of the nucleic acid, a wide range of cell types amenable to transfection, a high efficiency of transfer, lack of immunogenicity of the complexes and availability of the cationic lipids through chemical synthesis (Farhood et al., Annals N.Y. Acad. Sci. 716:23-35, 1994).
Cationic lipid-mediated delivery of nucleic acid to a wide variety of cell types has been demonstrated in vitro and in vivo. For example, nucleic acid encoding the cystic fibrosis transmembrane conductance regulator (CFTR) complexed with cationic lipids has been delivered to mouse lungs by intratracheal installation (Yoshimura et al., Nucleic Acids Res. 20:3233-3240, 1992) or by aerosol delivery (Stribling et al., Proc. Natl. Acad. Sci. 89:11277-1281, 1992). The delivery of CFTR using cationic lipids to a mouse model of cystic fibrosis (CF) produced correction of the ion channel defect (Hyde et al., Nature 362:250-255, 1993). Human clinical studies with cationic lipid-mediated delivery of the CFTR gene to CF patients demonstrated expression of the gene in nasal epithelium and no adverse clinical effects (Caplen et al., Nature Medicine 1:39-46, 1995).
Systemic gene expression of a reporter gene following a single intravenous injection of a cationic lipid-DNA complex has been shown in mice (Zhu et al., Science 261: 209-211, 1993). Moreover, safety studies in rodents and non-human primates of systemically administered cationic lipid-nucleic acid complexes have shown no significant toxicity associated with administering such complexes (Parker et al., Human Gene Therapy 6:575-590, 1995).
Cationic lipid-mediated transfection of mRNA in tissue culture was demonstrated to lead to translation of the transcript (Malone et al., Proc. Natl. Acad. Sci. 86:6077-6081, 1989). Delivery of antisense oligonucleotides to human endothelial cells using cationic lipids provided increased cellular uptake of the oligonucleotides and increased activity thereof in the cells (Bennett et al., Mol. Pharm. 41:1023-1033, 1992). The use of cationic lipids complexed to retroviral particles has allowed viral infection of cells which lacked the appropriate virus receptor (Innes et al., J. Virol. 64:957-962, 1990) or enhanced retroviral transduction using complexes known as virosomes (Hodgson et al., Nature Biotechnol. 14:339-342, 1996).
Immunotherapy for cancer using cationic lipid-nucleic acid complexes containing major histocompatibility (MHC) genes directly injected into mice tumors elicited immune responses that resulted in tumor regression (Plautz et al., Proc. Natl. Acad. Sci. 90:4645-4649, 1993).
Human clinical studies are currently underway using cationic lipid-mediated delivery of DNA sequences encoding immunotherapeutic molecules in human melanoma, colorectal and renal cancer patients (Nabel et al., Proc. Natl. Acad. Sci. 90:11307-11311, 1993; Crystal, Science 270:404-410, 1995).
The cationic lipid formulations to date have often incorporated the phospholipid dioleoylphosphatidylethanolamine (DOPE). This phospholipid is thought to disrupt the endosomal membrane by destabilizing its bilayer structure, allowing the lipid-nucleic acid complex to escape endosomal degradation and to enter into the cytoplasm (Farhood et al., Biochem. Biophys. Acta 1235:289-295, 1995).
However, a significant obstacle in the widespread use of cationic lipid complexes for nucleic acid delivery to cells is the tendency of the complexes to form large aggregates in solution.